Liver cancer is the fifth most common malignant tumor worldwide (Ho, BioDrugs 25:275-284, 2011), with hepatocellular carcinoma (HCC) being the most common form. Cholangiocarcinoma (CCA) is another major form of primary liver cancer. Although surgical resection offers a standard method for treatment of the disease, only a small portion of patients are eligible for the procedure. Liver cancer does not respond to most chemotherapy drugs. Thus, there is a critical need for novel immunotherapy, such as antibody therapy. It has been suggested that glypican-3 (GPC3) represents an attractive target for liver cancer therapy given that it is highly expressed in HCC (Capurro et al., Gastroenterology 125:89-97, 2003; Capurro and Filmus, Cancer Res 65:372, 2005; Ho and Kim, Eur J Cancer 47:333-338, 2011; Allegretta and Filmus, Anticancer Agents Med Chem 11:543-548, 2011).
The GPC3 gene encodes a 70-kDa precursor core protein, which can be cleaved by furin to generate a 40-kDa amino (N) terminal protein and a 30-kDa membrane-bound carboxyl (C) terminal protein. The C-terminus is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. GPC3 has been suggested as a target for both antibody (Ishiguro et al., Cancer Res 68:9832-9838, 2008; Nakano et al., Biochem Biophys Res Commun 378:279-284, 2009; Nakano et al., Anticancer Drugs 21:907-916, 2010) and cell-based (Nakatsura et al., Clin Cancer Res 10:8630-8640, 2004; Komori et al., Clin Cancer Res 12:2689-2697, 2006) immunotherapies. However, GPC3 expression is highly heterogeneous in HCC and other cancers (e.g., ovarian clear cell carcinoma and melanoma) (Suzuki et al., Cancer Sci, 102:1622-1629, 2011). Ideal therapeutic monoclonal antibodies (mAbs) should eliminate tumor cells expressing low levels of target antigen. Research in the area has been hampered by the lack of high-affinity mAbs that could be used to detect low expression of GPC3 in tumor cells for cancer therapy and diagnostics.
Filmus and colleagues developed the widely-used 1G12 mAb specific for the C-terminus of GPC3 and established an ELISA method to detect serum GPC3 in HCC patients (Capurro et al., Gastroenterology 125:89-97, 2003). Hippo et al. developed mAbs specific for the N-terminus of GPC3 (Hippo et al., Cancer Res, 64:2418-2423, 2004). While both studies detected soluble GPC3 protein in HCC culture supernatant or in the circulating blood of cancer patients, it is not clear whether the N-terminal or C-terminal subunit actually represents the soluble GPC3 format (Capurro and Filmus, Cancer Res 65:372, 2005). Prior studies indicate that concentrations of serum GPC3 are generally not high in patients and that none of the readily available mAbs can be used to measure serum GPC3, most likely due to low affinity of the mAb for GPC3.